Characterization of Citrus Tristeza Virus Isolates by Single-Strand Conformation Polymorphism Analysis of DNA Complementary to Their RNA Population

Abstract

Citrus tristeza virus (CTV) isolates, like other RNA viruses, contain a population of genomic variants, the composition of which may affect their biological characteristics. Analysis of single-strand conformation polymorphism (SSCP) is a simple technique that allows detection of minor variations in the nucleotide sequence of DNA fragments without sequencing. To assess if SSCP could be used to characterize the population of sequence variants present in CTV isolates, dsRNA from two isolates, differing by their SSCP profile of the gene p20, was combined at various ratios and the mixtures used as templates to synthesize cDNA by RT-PCR. The SSCP profile obtained from each dsRNA mix was a composite of the individual profiles of the two isolates, and the intensities of the cDNA bands reflected the relative proportion of each isolate in the dsRNA mix. The SSCP pattern of each component could be observed in the composite pattern when its dsRNA was in a ratio of at least 10%. Graft or slash-inoculation of CTV isolate T317 from citron to sweet orange, or from citrus to lime and then to sweet orange, caused changes in its SSCP profile, indicating host selection of the predominant sequence variants in the population. In some cases, this change was reversed when the isolate was back inoculated into the first host species, whereas in others the change was permanent. Variations in the viral population after change of host may help to explain biological diversity observed among CTV isolates.